The discovery of RNA interference (RNAi) and its potential to silence any gene of interest via the RNA-induced silencing complex (RISC) has revolutionized modern genetics and launched a new class of therapeutics termed short interfering RNA (siRNA). The first clinical trials of siRNA were initiated in patients with choroidal neovascularization (CNV) due to age-related macular degeneration (AMD), which is the leading cause of blindness among the elderly in industrialized world and afflicts more people in the United States than all cancers combined. In new and exciting recent work, we made the surprising discovery that siRNAs uniformly suppress experimental CNV in mice, independent of RNAi. We found that that siRNAs against non- mammalian genes, genes not expressed in the eye, or random sequences without homology to any known gene, all suppressed CNV, as did a modified siRNA incapable of RISC incorporation. This novel class effect was mediated via activation of toll like receptor-3 (TLR3) and its adaptor protein TRIF, and induction of interferon-3 and interleukin-12. Interestingly, siRNAs targeting vascular endothelial growth factor (VEGF)-A or its receptor VEGFR-1, which are in Phase II clinical trials, also suppressed CNV via TLR3 activation and not cognate transcript knockdown. We will test the hypothesis that both targeted and non-targeted siRNAs activate TLR3 by direct interaction and trigger a cellular program of vascular growth suppression. The specific aims of this proposal are to further define the interaction between siRNA and TLR3, determine the precise anti-angiogenic mechanisms of siRNA-induced CNV suppression in mice, and test the efficacy of non-targeted siRNA in a non-human primate model of CNV. These studies will provide clarifying insights into the mechanisms underlying unanticipated siRNA activities in vivo, define the molecular architecture governing this intersection of vascular biology and innate immunity, provide pharmacogenetic data that will enable personalized medicine in patients with TLR3 mutations, and propel the development of new, more affordable anti-angiogenic therapeutics for the public health epidemic that is AMD.Angiogenesis (the growth of abnormal blood vessels) is responsible for the majority of blindness in patients with age-related macular degeneration (AMD), which accounts for a lion's share of the annual $51 billion cost of blindness in the United States. The Nobel Prize winning discovery of RNA interference led to the development of short interfering RNA (siRNA) as a new class of targeted drugs, which were first launched in clinical trials in AMD. Public Health Relevance: Our surprising discovery that all siRNAs have intrinsic anti-angiogenic activity has immediate and profound implications both for ongoing clinical trials and for treating the estimated 500 million people with diseases driven by angiogenesis.